Further kinetic characterization of the non-allosteric phosphofructokinase from Escherichia coli K-12.

The labile non-allosteric form of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to a specific activity of 107 U/mg (2078-fold) from aerobic cultures of Escherichia coli K-12. The enzyme has an isoelectric point (pI) of 5.1, a native molecular weight of 67 000 +/- 3000 and a subunit weight of 34 000 +/- 400. A number of divalent metal ions can substitute for Mg2+ in the enzyme reaction in decreasing order Mn2+ > Mg2+ > Co2+ > Ca2+. In the presence of excess Mg2+, nucleotides do not affect the Km for fructose 6-phosphate with a value of 0.042 mM. The order of efficiency for nucleotides to act as phosphoryl donors is ATP > ITP > GTP > UTP > CTP. This remains unchanged in the presence of excess Mn2+, but V is increased 2.4-fold with ATP. A 2 : 1 ratio of Mn2+/nucleotide 5'-triphosphate produced an equivalent dissociation constant of 1.1 mM for all nucleotides, which was markedly decreased at a high Mn2+ level. The rate of enzyme catalysis was found to be dependent on the concentration of MnATP2-. Mn2+ at non-limiting values does affect the binding of fructose 6-phosphate to the enzyme.

Microbial cultures in the utilization of cellulosic materials.

This review elaborates on the most recent microbial development in saccharification of cellulose and cellulase formation. A particular highlight is a new genetic-immunochemical approach investigating the mechanism of adhesion of bacterial cellulase to cellulose during cellulose conversion. New developments and recent reviews in hemicellulose and lignin degradation are also covered.

Operational parameters for packed beds in solid-state cultivation.

Packed bed cultivation systems have potential for widespread application in solid-state cultivation (SSC), but they are poorly characterized. The effects of particle size and substrate loading on the growth of Rhizopus oligosporus on sago-beads in packed bed bioreactors were investigated. Pressure drop and protein were monitored as indicators of fungal growth in cultivations performed in a large column (4.9 cm internal diameter and 60 cm height) and a system of small columns (4.2 cm internal diameter and 5.2 cm height). The differential pressure drop increased to a maximum between 34 and 44 h and then decreased again. The maximum differential pressure drop attained was greatest for the smallest particle size and for the lower substrate loadings. However, since the protein content continued to increase throughout the cultivation, pressure drop could not be used to monitor growth directly.

Laboratory and clinical evaluation of two glucose meters for the neonatal intensive care unit.

OBJECTIVE: To evaluate the analytical and clinical performance of the One Touch II and Advantage glucose meters for use in neonatal specimens. DESIGN AND METHODS: For the laboratory evaluation, a total of 96 umbilical cord whole blood specimens were analyzed on the One Touch II and/or Advantage meters. Samples were centrifuged after analysis on the meters and plasma glucose was determined on the Hitachi 917. For the clinical evaluation, a total of 64 infants had specimens analyzed on each of the meters as well as on the laboratory analyzer. RESULTS: In the laboratory and clinical evaluations, both meters correlated well (r > 0.9, p < 0.001) with the plasma values for the Hitachi 917. However, the mean difference between the Advantage and Hitachi 917 was lower than that of the One Touch II in both the laboratory (-0.23 vs -0.64 mmol/L) and the clinical evaluations (-0.08 vs -0.60 mmol/L). 53.1% of One Touch and 26.6% of Advantage results from the clinical study had a discrepancy of > 0.5 mmol/L from the laboratory value. CONCLUSIONS: For neonatal specimens, glucose meters must have good low end precision, sensitivity and accuracy, In this study, the Advantage meter had fewer discordant results and better correlation with the Hitachi 917. Overall, nursing and laboratory staff preferred the performance and characteristics of the Advantage meter.

Joint venture capital investment for clean technologies and their problems in developing countries.

All technological developments are aimed at improving the quality of life of a community of people. Biotechnology is a technology which allows the exploitation of microorganisms, plants and animal cells to take place within an economic framework. Developing countries are looking for programmes achieving sustainable, economical growth conducive to a higher per capita income of the community. Any joint venture which promises social advances and economic benefits will have to be rural-based. This presentation discusses the need for a change in fermentation industry attitudes to allow joint venture capital investment in clean technologies together with the problems developing countries face for the implementation of such technologies.

ATP-sensitive and ATP-insensitive phosphofructokinase in Escherichia coli K-12.

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150,000 (subunit mol. wt 73,000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350,000 (subunit mol. wt 90,000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

Influence of carboxylic acids on the stereospecific nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases of Leuconostoc mesenteroides.

Leuconostoc mesenteroides increased its lactic acid production from glucose threefold when malic acid was added to the culture. This increase resulted also in a reduction of the ratio of d-lactic acid to l-lactic acid (31.5 to 1.23). Addition of malic acid increased 6.5-fold the specific activity of nicotinamide adenine dinucleotide (NAD)-linked l-lactate dehydrogenase and increased 3.2-fold that of NAD-linked d-lactate dehydrogenase. The Michaelis constant (K(m)) for NAD of the NAD-linked l-lactate dehydrogenase increased with the addition of malate, but no change was observed in the K(m) values for the respective d-enzyme. The effect of carboxylic acids on the NAD-linked l-lactate dehydrogenase activities was tested by using partially purified enzyme preparations from cells grown with glucose alone and from cells grown with glucose plus malate. Malate stimulated the l-enzyme and inhibited the d-lactate dehydrogenase. The NAD-linked l-lactate dehydrogenase exhibited the same activity bands on polyacrylamide gel electrophoresis whether the cell-free preparation originated from cells grown on glucose plus malate or on glucose as the sole carbon source. The NAD-linked d-lactate dehydrogenase, however, exhibited a different pattern of electrophoretic mobility, depending upon the source of origin of the cell-free preparation. The results suggest that malate has a stimulatory effect on the synthesis of both enzymes and may result in rearrangement of the protein structure of the d-lactate dehydrogenase. This rearrangement apparently makes the d-enzyme more susceptible to inhibition of catalytic activity. The l-lactate dehydrogenase, however, is stimulated not only in its synthesis but also in its activity. It is proposed that these effects are responsible for the regulation of lactic acid production.

Changes in the growth and enzyme level of Zymomonas mobilis under oxygen-limited conditions at low glucose concentration.

Zymomonas mobilis growing aerobically with 20 g glucose-1 (carbon-limited) in a chemostat exhibited an increase in both the molar growth yield (Yx/s) and the maximum molar growth yield (Yx/smax) and a decrease in both the specific substrate consumption rate (qs) and the maintenance energy consumption rate (me). Stepwise increase in the input oxygen partial pressure showed that anaerobic-to-aerobic transitional adaptation occurred in four stages: anaerobic (0 mm HgO2), oxygen-limited (7.6- 230 mm HgO2), intermediate (273 mm HgO2), and oxygen excess (290 mm HgO2). The steady-state biomass concentration, Yx/s, and intracellular ATP content increased between oxygen partial pressures of 7.6 and 120 mm HgO2, accompanied by a decrease in the qs and the specific acid production rate. The membrane ATPase activity decreased with increasing oxygen partial pressure and reached its lowest levels at 273 mm HgO2, which was the highest input oxygen partial pressure where steady-state conditions were possible. Glucokinase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase activities also decreased when the oxygen partial pressure was increased above 15 mm Hg, whereas pyruvate decarboxylase was unaffected by aeration. Growth inhibition at 290 mm HgO2 was characterised by a drastic reduction in the pyruvate kinase activity and a collapse in the intracellular ATP pool. The growth and enzyme data suggest that at low glucose concentrations and oxygen-limited conditions, the increase in biomass yields is a reflection of a redirection of ATP usage rather than a net increase in energy production.

Optimization of physiological factors for direct saccharification of cassava starch to glucose by Rhizopus oligosporus 145f.

Direct saccharification of 2.64% cassava starch by Rhizopus oligosporus 145F was attempted under various cultural conditions. Maximum glucose yield of 18.0 g/L culture filtrate was obtained with an initial pH 3.8, 2% (v/v) inoculum of R. oligosporus spores, and an incubation temperature of 45 degrees C in shake flask cultures for 48 h. This concomitantly produced 2.7 g mycelia/100g cassava starch containing 20.2% true protein. The production of glucose and mycelia was accomplished with 92.8% starch saccharification having 67.9% starch to glucose conversion efficiency.

Use of Cibacron Blue--Sepharose 6B to detect modifications of the non-allosteric 6-phosphofructokinase from Escherichia coli K-12.

In crude cell-free extracts of aerobically grown E. coli K-12, the non-allosteric form of 6-phosphofructokinase has a tetrameric molecular weight 140 000 with a low affinity (less than 5%) for the blue dextran chromophore--Cibacron Blue. The allosteric form has the same tetrameric molecular weight, but possesses a strong affinity for the blue dextran chromophore. Under conditions of prolonged storage, purification procedures of mild heat treatment (50 degrees C), the non-allosteric form converts to an active dimer (mol. wt 67 000), which binds to Cibacron Blue (less than 90%). Acid precipitation plus heat treatment prevents the conversion to the dimeric form and retains low Cibacron Blue affinity. These results are consistent with the isolation of a low molecular weight form and suggest that the inherent lability of this enzyme might be due to both non-specific proteolytic modification and a weak quaternary structure.

Transitional steady-state investigations during aerobic-anaerobic transition of glucose utilization by Escherichia coli K-12.

Transitional steady-state investigations during changes in oxygen tension under aerobic and during aerobic-anaerobic transition conditions were carried out with the aim of finding an indicator system which separates the equilibrium from the non-equilibrium state. Of the parameters used i.e. biomass formation, CO2 production, Q02, NADH oxidase, succinate dehydrogenase, phosphofructokinase, glyceraldehyde-3 phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and 2-oxoglutarate dehydrogenase, only the three enzymes requiring NADH or NADP for their function fulfilled the requirements. Biomass production and CO2 formation were useful only during the aerobic-anaerobic transition period. In each case the response was immediate and the indicator systems demonstrated that a new steady state of oxygen was always obtained after 11 h which, at the specific growth rate used, was equivalent to at least two volume replacements of the growth vessel.

Bioenergetic aspects of aerobic glucose metabolism of Escherichia coli K-12 under varying specific growth rates and glucose concentrations.

An attempt was made to find a bioenergetical explanation for the differential effect of specific growth rate and glucose concentration on glucose metabolism of Escherichia coli K-12 with the help of 2,4-dinitrophenol (DNP). The effect of DNP on biomass occurred only at high glucose concentrations. The presence of this uncoupler strongly stimulated glucose uptake rates and oxygen uptake rates, but repressed severly Yg values. Increase in glucose concentration, however, sharply decreased QO2. The amount of oxygen required for maintenance was not affected by DNP, but Yomax values were much lower in the presence of DNP. The results are discussed and it is suggested that aerobic fermentation is caused by a severe reduction of site 1 of the respiratory chain region, whereas biomass formation is affected by repression of the terminal cytochrome a2. In comparing the effect of glucose on biomass formation at similar Qglucose levels aerobic and anaerobic fermentation, repression occurred in both cases at glucose concentrations of 0.3% and above. Although the analyses of 15 enzymes established the metabolic differences, the repression of growth was common to both fermentation types.

Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.

Screening and characterization of bioflocculant produced by isolated Klebsiella sp.

Sixteen strains of polymer-producing bacteria were isolated from the activated sludge samples taken from two seafood processing plants in Southern Thailand. Their culture broths possessed the ability to flocculate kaolin suspension in the presence of 1% CaCl2. Based on the flocculating activity, the strain S11 was selected and identified to be a Klebsiella sp. using the partial 16S rRNA sequencing method. The growth of the isolated Klebsiella sp. was maximal (1.026 g l-1 dry cell mass) after 1 day cultivation while the highest polymer yield (0.973 g l-1) was achieved after 5 days cultivation. The flocculating activity of the culture broth, however, was highest after 2 days cultivation. The polymer was identified to be an acidic polysaccharide containing neutral sugar and uronic acid as its major and minor components, respectively. Results on the properties of the partially purified polysaccharide from Klebsiella sp. S11 revealed that it consisted of galactose, glucose and mannose in an approximate ratio of 5:2:1. It was soluble in acidic or basic solutions but not in organic solvents. Its molecular mass was greater than 2 x 10(6) Da. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl groups in its molecules. Differential scanning calorimetry of the polysaccharide indicated the crystalline melting point (Tm) at 314 degrees C. The optimum dosage of polysaccharide to give the highest flocculating activity was 15 mg l-1 in the presence of 1% CaCl2.

Effect of specific growth rate and glucose concentration on growth and glucose metabolism of Escherichia coli K-12.

Chemostat cultures of E. coli K-12 revealed that the metabolic change from respiration to aerobic fermentation can be obtained with increasing specific growth rate at low glucose input concentration (0.1%), or increasing glucose input concentrations at low specific growth rate (0.1 h-1). Both effects do not affect biomass formation. The metabolic change is not related to a pathway switch of glucose utilization. The increase in specific growth rate causes suppression of succinate dehydrogenase, and NADH oxidase, whereas glucose increases cause suppression of succinate dehydrogenase, cytochrome a and 2-ketoglutarate dehydrogenase. Both phenomena are reflected in the specific oxygen uptake rate, specific carbon dioxide production rate and respiratory quotient values. Growth limitation could be related to a maximal glucose uptake rate of the cell and thus constitutes an entirely different effect caused by high glucose input concentration.

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Investigation into pyruvate kinases from Escherichia coli K-12 grown under aerobic and anaerobic conditions.

Two forms of anaerobic Escherichia coli K-12 pyruvate kinase (EC 2.7.1.40) were separated by ammonium sulphate fractionations. Pyruvate kinases I is allosteric and pyruvate kinase II is non-allosteric to phosphoenolpyruvate. The addition of 1 mM FDP reversed the allostery to normal Michaelis-Menten kinetics. AMP had no effect, whereas 8 mM ATP completely inhibited the enzyme. The enzyme showed normal kinetics with ADP as substrate. Mg2+ and Mn2+ stimulated whereas Cu2+ severely inhibited the enzyme, which could be reversed by the addition of 1 mM FDP. Citrate, alpha-ketoglutarate, succinate, fumarate and alanine inhibited the enzyme, whereas phenylalanine had no effect. The allosteric pyruvate kinase from aerobic cultures was not only activated by FDP, but also by AMP. FDP changed Km and Vmax, whereas AMP influenced only the Km. During aerobic-anaerobic transition, pyruvate kinase synthesis increases and reaches a maximum under anaerobic conditions. The degree of FDP activation remains constant, but AMP activation is lost during transition. Aerobic cultures of E. coli K-12 grown on gluconeogenic substrates exhibited pyruvate kinase II activity (non-allosteric), which was stimulated by FDP and by AMP. It has been suggested that E. coli may have two types of pyruvate kinase II depending on the substrate and two types of pyruvate kinase I depending on oxygen tension in the medium.